Silvopastures integrate trees, forages, and livestock. Tall fescue, the dominant forage in much of the U.S., harbors an endophyte that produces toxic ergot alkaloids. Diluting the sward with other forages can reduce ergot alkaloid concentrations, but it is unknown how shade impacts alkaloid production and productivity of different forage mixtures. This study tested the effects of increasing shade and mixture complexity on sward yield, botanical composition, nutritive characteristics and ergot alkaloid concentrations. Slatted shade structures created 30, 50, and 70% shade compared to full sun. Three forage mixtures were evaluated (SIMPLE = tall fescue and white clover; INTERMEDIATE = SIMPLE + orchardgrass and red clover; and COMPLEX = INTERMEDIATE + Kentucky bluegrass, birdsfoot trefoil, and alfalfa). Fifty and 70% shade reduced yield while red clover and orchardgrass dominated shaded swards. Birdsfoot trefoil, Kentucky bluegrass, and white clover did not perform well in any treatment. Nutritive value declined beneath shade in spring and fall. Sward ergot alkaloid concentration increased beneath shade in simple mixtures because of greater proportions of tall fescue. In the intermediate and complex mixtures, ergot alkaloids were diluted by other forage species and was not affected by shade. This illustrates the importance of incorporating multiple species into the sward. Low light levels may not have been sufficient to meet the forages' high energetic demands in the spring. Even though total forage production or nutritive value may be sacrificed during part of the year, this may be compensated for by diverse swards diluting ergot alkaloid concentrations. / Master of Science

Two experiments were conducted at the University of Kentucky, Eden Shale Farm, Owenton, KY to evaluate the use of MTB-100TM (Alltech, Inc., Nicholasville, KY) to alleviate the symptoms of fescue toxicity when lactating Angus x Beefmaster cows and their calves grazed endophyte-infected KY-31 tall fescue. Experiment 1 provided a carbohydrate based toxin adsorbent, MTB-100TM, ad libitum in a commercial mineral supplement to project a daily consumption rate of 0, 20 or 40 g of MTB-100TM per cow. Cows were weighed, assigned a body condition score (BCS) and hair coat score (HC), rectal temperatures were recorded and fecal grab samples were taken for ergovaline (EV) and lysergic acid (LA) analysis every 35 days for three grazing seasons (May to September). Calves were also weighed and assigned a HC score. Although MTB-100TM did not improve cow or calf performance, cows older than 4 years and those with greater Beefmaster breeding exhibited a higher tolerance to fescue toxicity than 2 and 3-yr-olds and cows with greater Angus breeding. Experiment 2 was conducted to evaluate the response of lactating beef cows and their calves to strategic supplementation with MTB-100TM. MTB-100TM was mixed with a complete mineral so daily intake was projected to be 0 or 20 g/cow. The experimental period extended from May 5 to October 2 and was divided into 3 strategic periods: P1 = May 5 to July 5; P2 = July 5 to August 31; P3 = August 31 to October 2. Treatments were either 0 or 20 g•cow-1•d-1 MTB-100TM within a period (Treatment 1 = 0, 0, 0; Treatment 2 = 20, 0, 20; Treatment 3 = 0, 20, 0; Treatment 4 = 20, 20, 0; and Treatment 5 = 20, 20, 20). Cow and calf performance was measured the same as Exp. 1, but every 21 days. Supplementation early in the grazing season tended to improve cow weight gain and body condition; however, there was no effect on calf performance. Fecal output of EV and LA did not increase in either experiment with supplementation. In conclusion, strategically invoked MTB-100TM consumption can increase performance of cows grazing endophyte-infected tall fescue forage.

Graduation date: 1998

Endophytic fungi (A. coenophialum and A. lolii) which infect grasses produce ergot alkaloids that serve as the grasses' chemical defenses and enhance the vigor of the grass. Turf-type tall fescue with high endophyte levels has been deliberately developed to produce a greener, more vigorous, pest-resistant turf. Consumption of endophyte-infected grass causes various toxicity symptoms in livestock. Cattle in the southeastern and midwestern United States, where tall fescue is grown on 14 million hectares, often develop signs of toxicosis during summer months from grazing plants in fected by A. coenophialum. A more severe form of the disease, fescue foot, has been associated with cold environment and reported in late fall and winter months not only in the southeastern United States but also in the northwest United States. In New Zealand, where perennial ryegrass is grown on 7 million hectares of pasture, sheep often develop a condition called ryegrass staggers from grazing plants infected by A. lolii. New Zealand reports economic losses grazing plants infected by A. lolii. New Zealand reports economic losses associated with the sheep industry of $205 million per year. In the United States, economic losses associated with the beef cattle industry alone is estimated at $600 million per year. Range finding experiments and case studies of fescue foot and perennial ryegrass staggers (PRGS) were conducted on cattle and sheep under grazing and barn conditions. The main objective was to determine threshold levels of the endophyte toxins, ergovaline (EV) (appendix 1) and lolitrem B (appendix 2), associated with the diseases of fescue foot and PRGS respectively. Fescue foot was experimentally induced in cattle under barn studies in the spring with 825 ppb ergovaline. The ergovaline contaminated feed was given for a period of 42 days. Similar barn studies in sheep in spring to early summer did not produce clinical fescue foot with up to 1215 ppb. Field studies of natural fescue foot in a herd of sheep were conducted, (ie 540 ppb) values of ergovaline in the feed, but clinical disease was not produced in late fall through winter. A case study from a herd of sheep revealed 813 ppb dietary ergovaline had produced fescue foot in the months of fall (November). Fields of perennial ryegrass (PRG) where sheep received 2,135 ppb lolitrem B toxin were associated with clinical cases of PRGS in 42 sheep of 237 sheep (18 percent incidence rate) in the Willamette Valley of Oregon. Three months later, sheep on this same field which then had 1,465 ppb lolitrem B, did not have PRGS. These were the first range finding experiments undertaken in this locale to document threshold levels of endophyte toxins associated with fescue foot and PRGS. / Graduation date: 1994

Twenty-four steers were grazed on endophyte (Neotyphodium coenophialum)-infected [[Lolium arundinaceum (Schreb.) Darbysh]; E+] tall fescue (TF) and exposed to ergot alkaloids for an 106-d grazing period. Cattle were removed from pasture, placed in dry lots, and fed a non-toxic diet to evaluate changes in vascular contraction relative to time-off E+ TF pasture. Lateral saphenous veins (SV) were biopsied from steers at 0-, 21-, 42-, and 63-d off TF pasture and from 6 control steers at 0- and 63-d off bermudagrass (BG) pasture. To evaluate contractile response, biopsied SV were exposed to increasing concentrations of ergotamine in a multimyograph. Cross-sectional scans of the caudal artery were taken using color Doppler ultrasonography on 0-, 8-, 15-, 21-, 29-, 36-, 42-, and 45-d to determine artery luminal area. Contractility of SV was less for TF than BG steers on d 0, but was similar between the two treatments by d 63. Luminal areas of the caudal arteries in E+ TF steers relaxed over time and were similar to BG steers by 36 d off pastures. Results indicated that alkaloid-induced vasoconstriction in cattle grazed on E+ TF can be relaxed in 5 to 6 weeks after they are placed on non-toxic diets.

Tall fescue (Festuca arundinacea Schreb.) is often infected by the endophytic fungus, Acremonium coenophialum (Morgan-Jones and Gams) The fungus produces ergo peptide alkaloids, especially ergovaline. Consumption of endophyte-infected (ED fescue forage by cattle decreases serum prolactin and average daily weight gains, which results in economic losses for producers. Anecdotal reports suggest cattle with fescue toxicosis may not respond to vaccination. Hyperprolactinemia decreases cell-mediated and humoral immune responses in mice. Therefore, steers grazing EI or endophyte-free (EF) fescue forages were vaccinated to assess humoral immune responses. Steers grazing EI fescue exhibited classical clinical signs of fescue toxicosis, and mounted humoral immune responses to vaccination, despite hyperprolactinemia. Lymphocyte proliferation responses to mitogens in mice fed EI diets were similar to mice fed EF diets. Production of interferon gamma and interleukin-4 was higher by splenocytes from mice fed EI diets, whereas interleukin-2 tended to be lower. Fescue toxicosis may stimulate T helper cell 2 subset of CD4⁺ T cells. The T_H2 subset may augment humoral immune responses to vaccination. / Ph. D.

We conducted two experiments evaluating perennial ryegrass straw as a forage source for ruminants. Experiment 1 evaluated digestion and physiological variables in steers offered perennial ryegrass straw containing increasing levels of lolitrem B. Sixteen ruminally cannulated Angus X Hereford steers (231 ± 2 kg BW) were blocked by weight and assigned randomly to one of four treatments (TRT). Steers were provided perennial ryegrass straw at 120% of the previous 5-d average intake. Prior to straw feeding, soybean meal (SBM) was provided (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Low (L) and high (H) lolitrem B straws (<100 and 1550 ppb, respectively) were used to formulate TRT diets: LOW (100% L); LOW MIX (67% L:33% H); HIGH MIX (33% L:67% H); HIGH (100% H). Intake and digestibility of DM and OM, and ruminal pH, total VFA, and NH₃-N were not affected by increasing lolitrem B concentration (P>0.13). Ruminal indigestible ADF (IADF) fill increased linearly (P=0.01) and IADF passage rate (%/h) decreased linearly (P=0.04) as lolitrem B level increased. Experiment 2 evaluated performance and production of 72 Angus X Hereford cows (539 ± 5 kg BW) consuming perennial ryegrass straw containing increasing levels of lolitrem B during the last third of gestation. Cows were blocked by body condition score (BCS) and randomly assigned to one of three TRT. Cows were provided perennial ryegrass straw ad libitum and supplemented with SBM (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Mixtures of a L and H lolitrem B straw (467 and 2017 ppb, respectively) were used to formulate TRT diets: LOW (100% L); MIX (50% L:50% H); HIGH (100% H). Thirteen of 24 cows on the HIGH TRT exhibited signs of ryegrass staggers and were removed from the study. Dry matter intake was not affected (P>0.12) by increasing lolitrem B concentration; however, estimated DM digestibility decreased linearly (P<0.01) as lolitrem B concentration increased. Lolitrem B concentration did not influence pre- or post calving weight or BCS change (P>0.10). These data suggest that feeding perennial ryegrass straw containing up to 1550 ppb lolitrem B does not adversely affect nutrient digestion or physiological response variables in steers. However, providing straw with a lolitrem B concentration of approximately 2000 ppb resulted in 54% of cows exhibiting signs of ryegrass staggers. Blending of H and L straws appears to be a successful management practice. Keywords: Alkaloid, Beef Cattle, Endophyte, Lolitrem B, Perennial Ryegrass, Straw / Graduation date: 2004

Ergotalkaloide sind sekundäre Stoffwechselprodukte des parasitären Schlauchpilzes Claviceps purpurea der auf Getreide Mutterkörner (Sklerotien) bildet. In Sklerotien sind toxische Ergotalkaloide enthalten. Durch C. purpurea werden vorrangig sechs verschiedene Ergotalkaloid-Epimerenpaare gebildet, die toxischen C8-(R)-Epimere und die biologisch nicht relevanten C8-(S)-Epimere, die ineinander umgewandelt werden können. Das Ziel der Arbeit war es, die Epimerisierung der Ergotalkaloide während der Probenvorbereitung im Vergleich zu bisher bekannten Probenaufarbeitungsverfahren zu minimieren. Dieses gelang durch den Verzicht des Zusatzes starker Säuren oder Basen. Die aufgereinigten Extrakte können bei Raumtemperatur über 96 Stunden epimerisierungsfrei in einer tensidischen Acetonitril-Wasser-Lösung gelagert werden. Die Probenaufarbeitung mit anschließender Auftrennung über die Hochleistungsflüssigchromatographie und fluorimetrischer Detektion (HPLC-FLD) wurde für Roggenmehl und Speiseöl validiert und auf diese Martices angewendet. So konnten erstmals die Ergotalkaloidgehalte auch in Weizenkeimöl quantifiziert werden. Im zweiten Teil der Arbeit wurde das Epimerisierungsverhalten von Ergotalkaloiden bei Backversuchen und in vitro Verdauexperimenten untersucht. Das Backen resultierte in eine Verschiebung des Epimerengleichgewichtes auf die Seite der (S)-Epimere. Das angewendete in vitro Verdaumodell führte für die Ergotalkaloidepimerenpaare Ergotamin und Ergosin zu einer Verschiebung des Epimerengleichgewichtes auf die Seite der toxischen (R)-Epimere. Dagegen zeigten die Ergotalkaloide der Ergotoxingruppe eine Verschiebung des Epimerengleichgewichtes auf die Seite der (S)-Epimere. Der dritte Teil der Arbeit beschäftigt sich mit Ergotalkaloid-Konjugaten, die unter dem Einfluss von UV-Licht entstehen. Es wurden sechs Ergotalkaloid-Fettsäure-Konjugate synthetisiert und in Sklerotien über die HPLC in Verbindung mit massenspektrometrischer Detektion nachgewiesen. / Ergot alkaloids are secondary metabolites of the parasitic fungus Claviceps purpurea that forms sclerotia on cereals. These sclerotia contain toxic ergot alkaloids. C. purpurea forms six epimeric pairs of ergot alkaloids predominantly the toxic C8-(R)-epimers and the biologically inactive C8-(S)-epimers. In view of the fact that both epimeric forms can be transformed into one another, the objective of this work was to develop a novel sample preparation method that minimizes the epimerization rate compared to previously published methods. The presented sample preparation procedure minimizes epimerization of ergot alkaloids, as it operates without the addition of strong acidic or alkaline modifiers for matrix removal. After sample preparation, an ergot alkaloid containing extract in a sodium hexanesulfonate solution is obtained in which no epimerization after 96 hours was observed. Thus, the sample preparation allows extract storage at ambient temperature for prolonged HPLC analysis. This novel sample preparation followed by HPLC-flourescence analysis was validated for the matrices rye flour and wheat germ oil and was applied for food samples. This is the first time that the ergot alkaloid content in wheat germ oil was quantified. The second part of this work was the study of the epimerization behaviour of ergot alkaloids during baking and in vitro digestion. Baking of cookies resulted in a shift of the epimeric ratio towards the (S)-epimers. The in vitro digestion showed an ergot alkaloid specific shift of the epimeric ratio. The initial percentage of the (R)-epimer increased for ergotamine und ergosine. In contrast, ergot alkaloids of the ergotoxine type showed an epimeric shift towards their (S)-epimers. The third part of this work was the study of ergot alkaloid derivatives that are formed in combination with UV-light. Six different ergot alkaloid fatty acid derivates were synthesized and detected in sclerotia using a HPLC-MS/MS method.

Os alcalóides ergolínicos são uma categoria de compostos que possuem um esqueleto tetracíclico [6,5,6,6] derivados da ergolina. Do ponto de vista farmacológico, estes compostos são uma classe de produtos naturais importantes, já que exibem uma grande variedade de atividade biológica. Estas moléculas têm sido alvos sintéticos devido ao seu complexo esqueleto tetracíclico e as propriedades supracitadas, o que resultou no desenvolvimento de sínteses totais ao longo do tempo. O ácido lisérgico é o representante notável da família dos alcaloides ergolínicos, uma vez que desde o ano de 1956 até 2013 foram desenvolvidas treze sínteses totais do mesmo. Por isso, o nosso grupo de pesquisa propõe uma rota sintética para a construção de um intermediário indólico-piperidinico, que será usado para realizar a síntese total estereosseletiva do ácido lisérgico em um trabalho de pesquisa futuro. A estratégia para a síntese do intermediário baseia-se na reação de Horner-Wadsworth-Emmons (HWE) e na reação de inserção N-H intramolecular a partir de um aminoaldeído derivado do L-triptofano. A rota sintética inicia-se a partir da elaboração do reagente de olefinação de HWE em três etapas: reação de Michaelis Becker, hidrogenólise catalítica e formação do diazofosfonato. A construção do aminoaldeído requer cinco etapas: esterificação, proteção com Boc, desproteção seletiva, redução do éster e oxidação de Swern. O reagente de olefinação e o aminoaldeído reagem através da reação HWE, fornecendo a diazocetona &alpha;, &beta; -insaturada com configuração preferencial Z (Z:E=10:1). Finalmente a olefina Z reage mediante a reação de inserção N-H intramolecular, para fornecer o intermediário indólico-piperidínico. Adicionalmente, desenvolveu-se outra rota para construir o intermediário mencionado através da construção de um derivado de 4-nitroindol, porém esta não foi reprodutível. A síntese do intermediário indólico-piperidínico foi feita em sete etapas, partindo do L-triptofano com rendimento global de 14.9 %. / The ergot alkaloids are a class of compounds which have the tetracyclic skeleton [6,5,6,6] found in the ergoline molecule. These compounds are an important class of natural products that have wide biological activities. They have also been important synthetic targets due to their challenging tetracyclic skeleton as well as due to the previously mentioned biological properties. Lysergic acid is the main representative of the family of ergot alkaloids. Since 1956, thirteen total syntheses have been developed for this alkaloid. Considering the importance of lysergic acid and of the ergot alkaloids, our research group decided to propose a synthetic route to construct an advanced indole-piperidinic intermediate, which may be used to perform the total synthesis of lysergic acid and derivatives in a future work. The strategy to the synthesis of this advanced intermediate is based on the Horner-Wadsworth-Emmons reaction (HWE) and the intramolecular N-H insertion reaction, starting from a L-tryptophan aminoaldehyde derivative. The synthetic route started with the elaboration of the HWE olefination reagent in three steps: Michaelis Becker reaction, hydrogenolysis and diazophosphonate formation. In continuation, the construction of the aminoaldehyde required five steps: esterification, Boc protection, selective deprotection, ester reduction and Swern oxidation. The olefination reagent and the aminoaldehyde reacted by the HWE reaction furnishing an &alpha;, &beta; -unsaturated diazoketone with Z configuration (Z:E = 10:1). Finally the Z isomer reacted by means of an intramolecular N-H insertion reaction to provide the indole-piperidine intermediate. Additionally, we developed another route to construct a 4-nitro-indole intermediate, but this was not reproducible. The synthesis of the indole-piperidine intermediate was carried out in seven steps starting from L-tryptophan, with an overall yield of 14.9%.

Ergovaline is produced by the endophyte Neotyphodium coenophialum (Morgan-Jones and Gams) in tall fescue (Schedonorus arundinacea (Schreb.) Dumort. = Festuca arundinacea Schreb.) and is blamed for a multitude of costly livestock disorders. Testing of pastures is common in both research and on farm situations. Since ergovaline is known to be unstable and affected by many variables, the objective of this study was to determine the effect of sample handling and storage on the stability of this compound. Homogeneous milled tall fescue sub-samples were analyzed for ergovaline concentration using HPLC after a range of sample handling procedures or storage. Ergovaline was unstable in milled material after 24 hours in storage, regardless of temperature. The decrease in ergovaline after 24 hours ranged from 17 to 60%. These results show that tall fescue sample handling and storage have a significant effect on ergovaline concentrations. In conclusion, accurate laboratory analysis of ergovaline content may require that samples be transported immediately to the laboratory on ice for immediate analysis. Most laboratories are not equipped for same day analysis, therefore researchers and producers should acknowledge that laboratory ergovaline results may be lower than the actual content in the field.